Project 3A: To examine whether delta133p53-expressing, CD28-restored CD8+ T cells gain a cytotoxic activity on tumor cells in vitro, quantitative analyses of tumor cell survival and death is being performed in previously established experimental systems. For this purpose, we are collaborating with Dr. Carl June, University of Pennsylvania, a world-leading expert of chimeric antigen receptor (CAR)-T cell therapy. We have integrated the expression cassette of delta133p53 in Dr. June's CAR-T vectors and successfully generated CAR-T cells that express delta133p53. In a widely used B-ALL (B-cell acute lymphoblastic lymphoma) model co-cultured with CAR-T cells in vitro, delta133p53-expressing CAR-T cells showed longer-lasting and higher-efficient anti-tumor activity on B-ALL cells compared with currently standard CAR-T cells. In parallel to mechanistic studies on improved CAR-T effects by delta133p53, prompted by this promising co-culture result, we will move ahead to in vivo models and solid tumor models. Human tumor xenografts are established in immunodeficient mice by orthotopic or subcutaneous injection of human tumor cells, followed by intravenous injection of delta133p53-expressing or control CAR-T cells. The effects of delta133p53-expressing CAR-T cells on tumor growth will be evaluated by quantitative image analysis, mouse survival curve analysis, and apoptosis assays on tissue sections. If successful, this study will be a significant step towards future clinical application of delta133p53 in cancer immunotherapy. Project 3B: We have accumulated data from irradiated human astrocytes in vitro. We have found that delta133p53 expression is diminished upon radiation-induced senescence of astrocytes, along with increased SASP (e.g., IL-6). Lentiviral reconstitution of delta133p53 expression in astrocytes undergoing radiation-induced senescence has restored their proliferative potential and repressed SASP, and has upregulated RAD51 and enhanced DNA damage repair. Our in vivo data from tissue section analysis showed that irradiated brain regions obtained from melanoma patients with brain metastasis had increased numbers of p16INK4A-positive senescent astrocytes, while non-irradiated brain regions from the same patients, non-irradiated patients or non-disease controls did not. We have also applied our established expertise in astrocyte-neuron co-culture in vitro to show that delta133p53 can revert otherwise radiation-induced senescent, neurodegenerative astrocytes to neuroprotective ones, and that such phenotypic reversion of astrocytes involves repressed SASP and increased neurotrophic secretory signals from astrocytes to neurons. We are also performing experiments using chemotherapeutic drugs in similar experimental settings, including in vitro examination of the effect of delta133p53 on drug-induced astrocyte senescence and SASP, astrocyte-mediated neuronal death and survival in co-culture, and examination of brain tissues from cancer patients who were treated with chemotherapeutic drugs. Drugs that cross the blood-brain-barrier (BBB) and are used for treatment of brain tumors will also be examined for their direct effects on astrocytes and astrocyte-neuron interaction in culture. Project 3C: We plan to generate transgenic mice that express delta133p53 in an inducible manner. As an alternative model of the humanized expression of delta133p53 in mice, we have found that human p53 knock-in (Hupki) mice endogenously express human delta133p53 mRNA and protein. We have shown that the human delta133p53 in Hupki-derived cells is regulated via autophagic degradation as in human cells. We have also confirmed the expression of delta133p53 in Hupki brain and aorta tissues, which are the organs with the major pathological or fatal change in AD and HGPS, respectively. These delta133p53-humanized mouse models will be crossed with AD model mice (established by Dr. Bohr), whose isolated astrocytes in vitro have already been shown to be rescued from accelerated senescence by delta133p53. These mice will also be treated with brain radiation or chemotherapeutic drugs such as temozolomide, which induces cellular senescence in glioblastoma cells via downregulation of delta133p53. We also plan to enhance endogenous delta133p53 expression in Hupki mice by small molecule activators to be identified below in 3F. Project 3D: The shorter-than-normal replicative lifespan of HGPS patients-derived fibroblasts was significantly extended by increased expression of delta133p53 due to its dominant-negative inhibition of the senescence-inducing p53 target genes such as p21WAF1 and microRNA (miR)-34a. Delta133p53 also functioned to rescue the DNA repair defect characteristic of HGPS cells, marked by accumulated gamma-H2AX foci, via upregulating the DNA repair factor RAD51. Thus, a therapeutic value of delta133p53 in premature aging diseases is supported by its ability to correct the HGPS-associated cellular defects. To examine the effects of delta133p53 on shortened lifespan and accelerated aging-related pathologies in HGPS, we have crossed the Hupki mice with mouse models of HGPS and have been obtaining enough numbers of mice of different genotypes of Hupki and HGPS. Project 3E: Overexpression of delta133p53 extends the replicative lifespan of normal human cells but does not lead to cell immortalization, malignant transformation or chromosome abnormality. Normal human pluripotent stem cells, such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), express abundant levels of endogenous delta133p53. Enhanced expression of delta133p53 increases the efficiency of reprogramming from human fibroblasts to iPSCs that have normal chromosomes and form well-differentiated, benign teratomas, not malignant teratocarcinomas, which are induced by mutant p53. These delta133p53-inducible iPSC lines show lower rates of somatic mutations than iPSC under p53 knockdown. Whether these lower mutation rates are intrinsic to iPSC reprogramming or iPSC reprogramming is not by itself mutagenic, these data are strong evidence for no or minimal mutagenic activity of delta133p53. Thus our current data do not support the hypothesis that delta133p53 is oncogenic. Our recent gene expression profiling study using RNA-seq has further suggested that delta133p53 selectively represses senescence-inducing p53 target genes, which allows self-renewal of ESC/iPSC and validates its therapeutic values in senescence/aging-associated diseases, while delta133p53 does not interfere with the ability of p53 to repair DNA damage and eliminate severely damaged cells, which ensures genome stability of ESC/iPSC and minimizes a possibility of oncogenesis. Project 3F: We have shown that protein degradation via chaperone-assisted selective autophagy downregulates delta133p53 during cellular senescence in normal human cells such as astrocytes, fibroblasts and CD8+ T cells. This mechanistic study of delta133p53 provides an experimental basis for mechanism-oriented screening of small molecules. Towards development of p53 isoform-based therapies in both cancer and aging-associated diseases, we have been identifying small molecule compounds that upregulate the expression of delta133p53 protein, in particular, those that cross the BBB for astrocyte-mediated therapy in AD and radiation- and chemotherapy-induced brain damage. Using a cell-based, robust high-throughput screening strategy at NCATS, our initial screening of the LOPAC library has established the optimal conditions for screening, followed by screening of larger-size libraries and identification of candidate compounds, including a bromodomain protein-modulating compound that is known to enhance anti-tumor activity of T cells. These hit compounds will be tested in our established in vitro systems of cellular senescence and in Hupki mice in vivo.